Role of TGIF in Wnt-Induced Bone Formation

描述(申请人提供)：骨骼发育和内稳态依赖于一个更高层次的网络，该网络确保成骨细胞的骨形成和破骨细胞的吸收之间的有效平衡。这一网络的扰动往往与骨骼疾病有关，最严重的是骨质疏松症，具有固有的骨折风险以及相关的发病率和死亡率。骨重建的主要调节者是典型的Wnt信号通路，它严格控制成骨细胞分化的几个方面。同源结构域蛋白TGIF在转化生长因子-β信号通路中作为转录辅助抑制因子发挥作用，该信号通路被认为是限制骨形成的。为了促进我们对TGIF的生理功能和调控的理解，我们使用酵母双杂交试验筛选了TGIF结合蛋白。一种先前未知的引起我们注意的TGIF结合蛋白是Axin2，它通过介导破坏体的形成而发挥Wnt信号的负调节作用，这是一个信号模块，在这个信号模块中，蛋白激酶GSK3-？标记？-catenin进行泛素依赖的降解。在随后的功能获得和功能丧失研究中，TGIF被发现促进Wnt诱导的转录反应。我们的初步研究还表明，TGIF在Wnt信号诱导成骨细胞分化的能力中是必不可少的。值得注意的是，我们发现TGIF可以通过一种独立于其抑制转化生长因子-β信号转导能力的机制来调节骨形成，这揭示了TGIF的另一种生理功能是确保有效地决定成骨细胞对WNT的响应。与其在成骨细胞定位中的作用一致，去除小鼠的TGIF会损害成骨细胞的成熟并减少骨量。综上所述，这些发现导致我们提出了一个工作假说，即TGIF可能有助于调节骨骼中规范的Wnt信号，对破坏体支架复合体施加严格的控制。因此，本研究建议的具体目的是：目的1：进一步阐明TGIF影响Wnt介导的成骨细胞分化和骨形成的分子机制，特别是它在破坏小体的组装/拆解中的可能作用，从而-连环蛋白的稳定性。目的：分析TGIF基因的完全缺失和成骨细胞靶向缺失对骨内环境的影响，以及WNT在体内外诱导成骨细胞分化和成骨的作用。作为Wnt信号和骨形成调控的真正贡献者，TGIF的功能特征将加强我们对维持骨稳态的关键生理过程的了解，最终演变为设计和实施安全的合成代谢治疗药物来治疗骨质疏松症和其他低骨量综合征的新概念。 公共卫生相关性：WNT信号促进成骨细胞的增殖和分化，而该信号通路的干扰会影响骨骼的动态平衡，导致人类的骨骼疾病。我们发现，同源结构域蛋白TGIF的一个生理功能是确保在Wnt信号反应中适当地确定成骨细胞的命运。我们相信，他新发现的骨形成调节因子的功能表征将在了解成骨细胞及其骨基质分泌活动的调节机制方面开辟一个新的研究领域，并在药物发现方面开辟新的领域，希望改善骨质疏松症和其他低骨量综合征的治疗。

DESCRIPTION (provided by applicant): Skeletal development and homeostasis depend on a higher-order network that ensures efficient balance between bone formation by osteoblasts and resorption by osteoclasts. Perturbations of this network are often associated with skeletal disorders, with the most severe being osteoporosis, with inherent risk of fracture and associated morbidity and mortality. A master regulator of bone remodeling is the canonical Wnt signaling pathway, which exerts a tight control over several aspects of osteoblast differentiation. The homeodomain protein TGIF functions as a transcriptional corepressor in the transforming growth factor beta (TGF-¿) signaling pathway, which is known to restrict bone formation. To advance our understanding of how TGIF's physiological functions and regulation are controlled, we have screened for TGIF-binding proteins using the yeast two-hybrid assay. A previously unidentified TGIF binding protein that attracted our attention is Axin2, which functions as a negative regulator of Wnt signaling through its potential to mediate formation of the destructosome, a signaling module where the protein kinase GSK3-¿ marks ¿-Catenin for ubiquitin-dependent degradation. In subsequent gain- and loss-of-function studies, TGIF was found to promote Wnt-induced transcriptional responses. Our preliminary investigations also suggest that TGIF is essential to the ability of Wnt signaling to induce osteoblast differentiation. Strikingly, we found that TGIF can regulate bone formation by a mechanism independent of its ability to suppress TGF-¿ signaling, revealing that an alternative physiological function of TGIF is to ensure effective osteoblast cell fate determination in response to Wnt. Consistent with its role in osteoblast commitment, ablation of TGIF in mice impaired osteoblast maturation and decreased bone mass. Taken together, these findings led us to propose a working hypothesis in which TGIF may contribute to the regulation of canonical Wnt signaling in bone, imposing a stringent control over the destructosome scaffolding complex. Accordingly, the specific aims of this research proposal are: Aim 1: Further delineate the molecular mechanisms by which TGIF affects Wnt-mediated osteoblast differentiation and bone formation, with particular emphasis on its possible role in the assembly/disassembly of the destructosome, and thereby ¿-Catenin stability. Aim 2: Analyze the effects of full and osteoblast-targeted deletion of TGIF on bone homeostasis and the ability of Wnt to induce osteoblast differentiation and bone formation in vitro and in vivo. Functional characterization of TGIF as a bona fide contributor to the regulation of Wnt signaling and bone formation, will strengthen our knowledge of key physiological processes that maintain bone homeostasis, ultimately evolving into new concepts in the design and implementation of safe anabolic therapeutic drugs against osteoporosis and other low bone mass syndromes. PUBLIC HEALTH RELEVANCE: Wnt signaling fosters osteoblast proliferation and differentiation, and perturbations of this signaling pathway affect bone homeostasis, resulting in bone disorders in human. We discovered that a physiological function of the homeodomain protein TGIF is to ensure proper osteoblast cell fate determination in response to Wnt signaling. We believe that functional characterization of his newly discovered regulator of bone formation will open a new field of investigation both in terms of understanding the mechanisms that regulate osteoblasts and their bone matrix-secreting activity and in terms of drug discovery with the hope to improve the treatment of osteoporosis and other low bone mass syndromes.